ABSTRACT Retinal pigment epithelium (RPE) cells in the retina are the major site of disease pathogenesis in numerous macular degenerative diseases. In a subset of these maculopathies, the primary pathological manifestations are localized to the RPE-extracellular matrix (RPE-ECM) complex, suggesting that ECM abnormalities contribute to the underlying disease mechanisms and RPE dysfunction in these maculopathies. In fact, mutations in TIMP3, a gene involved in ECM regulation, leads to Sorsby's fundus dystrophy (SFD), an inherited maculopathy with phenotypic alterations that mimic age-related macular degeneration (AMD). In addition, rare variants of TIMP3 are strongly linked to AMD development and TIMP3 accumulates underneath the RPE in both SFD and AMD. Together, these findings suggest that TIMP3 biology plays a central role in maintaining RPE-ECM homeostasis and that TIMP3 dysfunction contributes to pathophysiology of some maculopathies. However, the consequence of TIMP3 dysfunction on RPE physiology and its role in the development of macular degeneration has not been established. In this proposal, we will elucidate the molecular and pathological consequences of TIMP3 mutation in RPE cells using a patient-derived human- induced pluripotent stem cell (hiPSC)-RPE model of SFD. In preliminary studies, SFD hiPSC-RPE develops key pathological manifestation of the disease including, drusen formation and accumulation of TIMP3 in the extracellular matrix. Furthermore, consistent with TIMP3's biological function and pathophysiology of maculopathies affecting RPE-ECM integrity, SFD hiPSC-RPE displays altered expression of 1) pro-angiogenic TIMP3-target matrix metalloproteinases (MMPs; MMP2, MMP14), 2) TNF converting enzyme (TACE) and 3) complement pathway genes. These results support the hypothesis that TIMP3 dysfunction in SFD leads to dysregulation of ECM-turnover and activation of pro-angiogenic and pro-inflammatory signaling that consequently leads to drusen formation and RPE-mediated CNV. To test this hypothesis, we will assess the following predictions in our model system 1)) increased TIMP3 accumulation in ECM in SFD alters ECM composition/turnover and instigates complement activation in turn promoting drusen formation, 2) TIMP3 dysfunction leads to increased expression/activity of MMPs, MMP2 and MMP14, and thereby impaired barrier integrity and VEGF release by RPE cells in SFD and 3) TIMP3 dysfunction leads to TNF-induced pro- inflammatory signaling by RPE. These proposed experiments will provide clear answers the specific role of TIMP3 mutation/dysfunction in drusen biogenesis and RPE-mediated CNV. Ultimately, the knowledge gained in this study will help identify potential drug therapies for targeting TIMP3-induced RPE-ECM alterations in SFD and other maculopathies.!